U.S. patent number 6,524,865 [Application Number 08/928,075] was granted by the patent office on 2003-02-25 for electrochemiluminescent enzyme immunoassay.
This patent grant is currently assigned to IGEN International, Inc.. Invention is credited to Pam Liang, Mark T. Martin, Rick Saul.
United States Patent |
6,524,865 |
Martin , et al. |
February 25, 2003 |
**Please see images for:
( Certificate of Correction ) ** |
Electrochemiluminescent enzyme immunoassay
Abstract
Electrochemiluminescent-labels and enzyme substrates, which
preferably are conjugated, are used in immunoassays and
electrochemiluminescence is generated catalytically. In
conventional electrochemiluminescence immunoassays, an anti-analyte
antibody molecule can give rise to typically 6-8
electrochemiluminescence-active ruthenium atoms, while in the
present invention, each enzyme-labeled anti-analyte molecule can
give rise to thousands of electrochemiluminescence-active ruthenium
atoms per second. An exemplary immunoassay is based on a catalytic
process employing .beta.-lactamase-conjugated anti-analytes which
enzymatically hydrolyze electrochemiluminescent-labeled substrates,
making them strongly electrochemiluminescent. The
electrochemiluminescence signal generated by each anti-analyte
molecule (i.e., each analyte molecule) is much greater than with
the conventional method. Accordingly, greater sensitivity can be
gained in the measurement of low concentrations of a given
immunoassay analyte.
Inventors: |
Martin; Mark T. (Bethesda,
MD), Saul; Rick (Gaithersburg, MD), Liang; Pam
(Arlington, VA) |
Assignee: |
IGEN International, Inc.
(Gaithersburg, MD)
|
Family
ID: |
23925520 |
Appl.
No.: |
08/928,075 |
Filed: |
September 11, 1997 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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484766 |
Jun 7, 1995 |
|
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Current U.S.
Class: |
436/172; 435/18;
435/7.6; 435/7.72; 435/7.9; 435/7.95; 436/501; 436/512;
436/513 |
Current CPC
Class: |
C07F
15/0053 (20130101); C07K 16/44 (20130101); G01N
33/533 (20130101); G01N 33/535 (20130101) |
Current International
Class: |
C07F
15/00 (20060101); C07K 16/44 (20060101); G01N
33/535 (20060101); G01N 33/533 (20060101); G01N
021/76 (); G01N 033/563 (); G01N 033/53 (); C12Q
001/34 () |
Field of
Search: |
;435/7.1,7.72,7.9,7.01,7.92,18,23,24,25,966,968,975,7.6,7.95
;436/172,501,512,513 ;252/700 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Chapter 11: "Inflammation" Section B: Enzyme-Linked Immunoabsorbent
Assay in Basic & Clinical Immunology, 8th Edition, 1994:
Appleton & Lange at p. 173-174 and see Figures therein.* .
Promega Catalog, p. 14.27.* .
Enzyme Nomenclature. Internation Union of biochemist and Molecular
Biologist. Academic Press, New York, New York, pp. v-xiii, 1992.*
.
Yang et al., "Electrochemiluminescence: A New Diagnostic and
Research Tool", 12 Bio/Technology 193-194 (2/94). .
Blackburn et al., "Electrochemiluminescence Detection for
Development of Immunoassays and DNA Probe . . . ", vol. 27, No. 9
Clin. Chem., 1534-1539 (1991). .
D.J. Payne, "Metallo-B-lactamases--a new therapeutic challenge", 39
J. Med. Microbiol. 93-99 (1993). .
S. Coulton and I. Francois, "6 B-Lactamases: Targets for Drug
Design", 31 Progress in Medicinal Chemistry, 297-349 (1994). .
Harold C. Neu, "The Crisis in Antibiotic Resistance", 257 Science,
1064-1072 (Aug. 21, 1992). .
A.C. Peterson et al., "Evaluation of four qualitative methods for
detection of B-lactamases . . . " vol. 8, No. 11 cur. J. Clin.
Microbiol. Infect. Dis., 962-967 (1989) (not currently available).
.
Yolken et al., "Rapid diagnosis of infections caused by
B-lactamase-producing bacteria . . . " vol. 97, No. 5 The Journal
of Pediatrics, 715-720 (11/80). .
S.C. Anderson and S. Cocayne, Clinical Chemistry: Concepts and
Applications, W.B. Saunders (1993) Philadelphia, PA (currently not
available). .
Yolken et al., "The Uses of Beta-Lacatamase in Enzyme Immunoassays
for Detection . . . " 73 J. Immunol. Meth., 109-123 (1984). .
Svensson et al., "Synthesis and Characterization of Monoclonal
Antibody-B-Lactamases Conjugates" 5 Bioconjugate Chem., 262-267
(1994)..
|
Primary Examiner: Venkat; Jyothsna
Assistant Examiner: Friend; Tomas
Attorney, Agent or Firm: Kramer Levin Naftalis & Frankel
LLP Evans, Esq.; Barry
Parent Case Text
This application is a continuation of application Ser. No.
08/484,766, filed Jun. 7, 1995 now abandoned.
Claims
We claim:
1. A method for detecting or quantitating an analyte in a sample
said method comprising: (a) contacting the sample with (i) a
binding partner of said analyte or a competitive binder which
competes with the analyte in a specific binding interaction, said
binding partner or said competitive binder being conjugated to an
enzyme; (ii) a substrate to said enzyme; and, (iii) an
electrochemiluminescent detectant, wherein said substrate and the
resulting product of said enzyme acting on said substrate differ in
their ability to generate electrochemiluminescence at an electrode
in the presence of said electrochemiluminescent detectant; (b)
imposing a voltage upon a working electrode in contact with said
sample and thereby causing said electrochemiluminescent detectant
to emit electrochemiluminescence; (c) detecting or measuring the
electrochemiluminescence emitted in step (b); and (d) comparing
said detection or measurement to a standard to determine whether or
in what amount said analyte is present in the sample.
2. A method as recited in claim 1 wherein said
electrochemiluminescent detectant is conjugated to said
substrate.
3. The method according to claim 2, wherein the enzyme is
.beta.-lactamase and the electrochemiluminescent detectant is
Ru(bpy).sub.3 +.sup.2 or an electrochemilunminescent derivative
thereof.
4. The method according to claim 1, wherein said substrate and said
electrochemiluminescent detectant are not conjugated to one
another.
5. The method according to claim 4, wherein the enzyme is
.beta.-lactamase and the electrochemiluminescent detectant is
Ru(bpy).sub.3 +.sup.2 or an electrochemiluminescent derivative
thereof.
6. A method for detecting or quantitating an analyte in a sample
said method comprising: (a) forming a complex comprising (i) said
analyte or a competitive binder which competes with the analyte in
a specific binding interaction, and (ii) a binding partner of said
analyte, said binding partner or said competitive binder being
conjugated to an enzyme; (b) contacting the complex with a
substrate to said enzyme and an electrochemiluminescent detectant;
(c) imposing a voltage upon a working electrode in contact with
said complex and thereby causing said electrochemiluminescent
detectant to emit electrochemiluminescence; (d) detecting or
measuring the electrochemiluminescence emitted in step (c); and (e)
comparing said detection or measurement to a standard to determine
whether or in what amount said analyte is present in the
sample.
7. The method of claim 6 wherein said substrate and said
electrochemiluminescent detectant are conjugated to one
another.
8. The method of claim 6 wherein said substrate and said
electrochemilunminescent label are not conjugated to one
another.
9. The method of claim 6, wherein the enzyme is .beta.-lactamase, a
protease or an oxido-reductase.
10. The method of claim 6, wherein the substrate is an antibiotic,
a peptide, or nicotinamide adenine dinucleotide.
11. The method of claim 6, wherein the electrochemiluminescent
detectant is selected from the group consisting of rubrene,
9,10-diphenyl anthracene, ruthenium containing compounds and osmium
containing compounds.
12. The method of claim 11, wherein the electrochemiluminescent
detectant is ruthenium II tris-bypyridine chelate.
13. The method of claim 7 wherein the electrochemiluminescence
generated in the presence of said electrochemiluminescence
detectant by the reaction product of the enzyme working on said
substrate/detectant conjugate is higher than the
electrochemiluminescence generated by the substrat/detectant
conjugate.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the development of an
electrochemiluminescence (ECL) based enzyme immunoassay for the
detection and the quantitative measurement of analytes. The
immunoassay is based on a catalytic process employing
.beta.-lactamase-conjugated anti-analytes which enzymatically
hydrolyze electrochemiluminescent substituted substrates, making
them strongly electrochemiluminescent. The immunoassay is very
sensitive and is suitable for the detection and monitoring of any
analyte for which an anti-analyte can be made.
2. Description of Related Art
An ever-expanding field of applications exists for rapid, highly
specific, sensitive, and accurate methods of detecting and
quantifying chemical, biochemical, and biological substances,
including enzymes such as may be found in biological samples.
Because the amount of a particular analyte of interest such as an
enzyme in a typical biological sample is often quite small,
analytical biochemists are engaged in ongoing efforts to improve
assay performance characteristics such as sensitivity.
One approach to improving assay sensitivity has involved amplifying
the signal produced by a detectable label associated with the
analyte of interest. In this regard, luminescent labels are of
interest. Such labels are known which can be made to luminesce
through photoluminescent, chemiluminescent, or
electrochemiluminescent techniques. "Photoluminescence" is the
process whereby a material luminesces subsequent to the absorption
by that material of light (alternatively termed electromagnetic
radiation or emr). Fluorescence and phosphorescence are two
different types of photoluminescence. "Chemiluminescent" processes
entail the creation of the luminescent species by a chemical
reaction. "Electrochemiluminescence" is the process whereby a
species luminesces upon the exposure of that species to
electrochemical energy in an appropriate surrounding chemical
environment.
The signal in each of these three luminescent techniques is capable
of very effective amplification (i.e., high gain) through the use
of known instruments (e.g., a photomultiplier tube or pmt) which
can respond on an individual photon by photon basis. However, the
manner in which the luminescent species is generated differs
greatly among and between photoluminescent, chemiluminescent, and
electrochemiluminescent processes. Moreover, these mechanistic
differences account for the substantial advantages as a
bioanalytical tool that electrochemiluminescence enjoys vis a vis
photoluminescence and chemiluminescence. Some of the advantages
possible with electrochemiluminescence include: (1) simpler, less
expensive instrumentation; (2) stable, nonhazardous labels; and (3)
increased assay performance characteristics such as lower detection
limits, higher signal to noise ratios, and lower background
levels.
As stated above, in the context of bioanalytical chemistry
measurement techniques, electrochemiluminescence enjoys significant
advantages over both photoluminescence and chemiluminescence.
Moreover, certain applications of ECL have been developed and
reported in the literature. U.S. Pat. Nos. 5,147,806, 5,068,808,
5,061,445, 5,296,191, 5,247,243, 5,221,605, 5,238,808 and
5,310,687, the disclosures of which are incorporated herein by
reference, detail certain methods, apparatuses, chemical moieties,
inventions, and associated advantages of ECL.
A particularly useful ECL system is described in a paper by Yang et
al., Bio/Technology, 12, pp. 193-194 (Feb. 1994). See also a paper
by Massey, Biomedical Products, October 1992 as well as U.S. Pat.
Nos. 5,235,808 and 5,310,687, the contents of these papers and
patents being incorporated herein by reference.
ECL processes have been demonstrated for many different molecules
by several different mechanisms. In Blackburn et al. (1991) Clin.
Chem. 37/9, pp. 1534-1539, the authors used the ECL reaction of
ruthenium (II) tris(bipyridyl), Ru(bpy).sub.3.sup.2+ are very
stable, water-soluble compounds that can be chemically modified
with reactive groups on one of the bipyridyl ligands to form
activated species with which proteins, haptens, and nucleic acids
are readily labeled.
Beta-lactamases which hydrolyze the amide bonds of the
.beta.-lactam ring of sensitive penicillins and cephalosporins are
widely distributed amongst microorganisms and play a role in
microbial resistance to .beta.-lactam antibiotics. Beta-lactamases
constitute a group of related enzymes which are elaborated by a
large number of bacterial species but not by mammalian tissues and
can vary in substrate specificities. See generally Payne, D. J., J.
Med. Micro (1993) 39, pp. 93-99; Coulton, S. & Francois, 1.,
Prog. Med. Chem. (1994) 31, 297-349; Moellering, R. C., Jr., J.
Antimicrob. Chemother. (1993) 31 (Suppl. A), pp. 1-8; and Neu, H.
C., Science (1992) 257, pp. 1064-1072.
Several methods currently exist for the detection of microbial
.beta.-lactamases. Some representative examples follow.
W. L. Baker, "Co-existence of .beta.-lactamase and penicillin
acylase in bacteria; detection and quantitative determination of
enzyme activities", J. Appl. Bacteriol. (1992) Vol. 73, No. 1, pp.
14-22 discloses a copper-reducing assay for the detection of
penicilloates and fluorescamine assay to detect 6-aminopenicillanic
acid concentrations when both substances were produced by the
action of the enzymes on a single substrate.
U.S. Pat. No. 5,264,346 discloses a calorimetric assay for
.beta.-lactamase which has a variety of applications. The assay is
based on the decolorization of a chromophore formed by oxidation of
either the N-alkyl derivative of p-phenylenediamine or the
3,3',5,5'-tetraalkyl derivative of benzidine. The decolorization is
attributed to the presence of an open .beta.-lactam ring product
resulting from the hydrolysis of cephalosporin or penicillin.
Decolorization with the open .beta.-lactam product of penicillin
requires the presence of a decolorization enhancer such as mercury
containing compounds. The enhancer is not required for
decolorization with the open .beta.-lactam product of
cephalosporin.
U.S. Pat. No. 4,470,459 discloses a rapid method for the detection
of the presence of .beta.-lactamase from microbial sources which is
based on a .beta.-lactamase conversion of a .beta.-lactam substrate
which reverses its ability to fluoresce. Specific .beta.-lactams
mentioned as having this property include ampicillin, cephalexin,
amoxicillin, cefadroxil and cephaloglycin. The change in the
ability to fluoresce is attributed to the presence of
.beta.-lactamase.
WO 84/03303 discloses a microbiological test process for
identifying producers of .beta.-lactamase. The assay relies on
changes in acidity which affect the fluorescence of the indicator
such as coumarin. This change in acidity is attributed to the
conversion product produced by the presence of the
.beta.-lactamase.
A. C. Peterson et al., "Evaluation of four qualitative methods for
detection of .beta.-lactamase production in Staphylococcus and
Micrococcus species", Eur. J. Clin. Microbiol. Infect. Dis. (1989),
Vol. 8, No. 11, pp. 962-7 presents certain factors which were
employed in evaluating qualitative assays for .beta.-lactamase.
Robert H. Yolken et al., "Rapid diagnosis of infections caused by
.beta.-lactamase-producing bacteria by means of an enzyme
radioisotopic assay", The Journal of Pediatrics, Vol. 97, No. 5
(November 1980) pp. 715-720 discloses a sensitive enzymatic
radioisotopic assay for the measurement of .beta.-lactamase as a
rapid test for detection of bacterial infection. The assay protocol
involves an incubation step with sample followed by the separation
step on a positively charged column such as DEAE-Sephacel prior to
measurement of the radioactivity of eluted fractions. The
.beta.-lactamase converted penicillinic product has an additional
carboxyl group which insures its stronger binding to the positively
charged column than the penicillin. Differences in radioactivity
between the eluted fractions and the original values are attributed
to the presence of .beta.-lactamase.
In immunoassays generally, antibodies (equivalently referred to
herein as "anti-analytes") are used to detect analyte. Commonly, an
anti-analyte is labeled with a molecule that is detectable by, for
example, absorbance, fluorescence, luminescence, or
electrochemiluminescence. Alternatively, the antibody can be
labeled with an enzyme that creates or destroys a compound with one
of these features. There are two main types of enzyme immunoassays;
enzyme-linked immunosorbant assays (ELISA) and enzyme-multiplied
immunoassay techniques (EMIT). S. C. Anderson & S. Cockayne,
Clinical Chemistry: Concepts and Applications, W. B. Saunders
(1993) Philadelphia, Pa. In enzyme immunoassays, the process is
catalytic such that multiple detectable labels are formed, giving
the possibility of enhanced sensitivity.
Electrochemiluminescence (ECL) immunoassays are conventionally
carried out with antibody conjugated to the label, which is
generally a derivative of tris(bipyridyl) ruthenium(II)
(abbreviated as Ru(bpy).sub.3.sup.2+) G. Blackburn et al. (1991)
Clin. Chem. 37, 1534-1539. In these assays, every antibody has a
limited number of Ru(bpy).sub.3.sup.2+ molecules on its surface
(for example, 6-8).
Compositions and methods have now been discovered for the
preparation and uses of .beta.-lactamase-conjugated antibodies in
ECL-based immunoassays. For example, the enzyme .beta.-lactamase
can efficiently hydrolyze Ru(bpy).sub.3.sup.2+ substituted
penicillins. The penicillins, termed Ru-Amp and Ru-APA, are only
very weakly electrochemiluminescent, but when they are hydrolyzed
by .beta.-lactamase according to the present invention they become
strongly electrochemiluminescent. The presence of .beta.-lactamase
therefore can be detected with a high level of sensitivity in an
ECL instrument using either of these compounds. As opposed to
conventional ECL immunoassays where the Ru(bpy).sub.3.sup.2+ label
is directly attached to the antibody, in the enzyme-based ECL
immunoassays of the present invention, the
electrochemiluminescently-active ruthenium complex is catalytically
generated by the enzyme attached to the antibody surface. Thus,
instead of one antibody permitting a few (typically 6-8) ruthenium
labels to generate light, one antibody-enzyme complex can generate
typically 2000 ruthenium labels per second and could generate as
many as 10,000 or more.
SUMMARY OF THE INVENTION
Conventional ECL-based immunoassays employ ruthenium labeled
antibodies. In the present invention, an immunoassay has been
discovered in which the ruthenium-labeled antibody is replaced with
an enzyme-labeled antibody. The enzyme is .beta.-lactamase.
Tripropylamine (TPA) or similar reductants are omitted from the
solution and, for example in the case of infection related assays,
ruthenium-labeled penicillins are used instead. In the presence of
.beta.-lactamase-labeled antibody, the ruthenium labeled substrates
are catalytically hydrolyzed, generating an enormous increase in
ECL. The assay is superior to the use of ruthenium-labeled antibody
immunoassays because enzyme-generated ECL-active ruthenium is a
catalytic process, forming many ECL active molecules.
Broadly stated, the invention contemplates an
electrochemiluminescence based immunoassay for the detection of
analytes. The invention employs enzymes such as .beta.-lactamases,
proteases or oxido-reductases conjugated to antibodies and ECL
labels and enzyme substrates, preferably ECL label substituted
substrates such as ECL label substituted antibiotics, peptides, and
nicotinamide adenine dinucleotide (NADH) which together provide an
antibody-enzyme complex which can catalytically generate up to
thousands of ECL active labels per second.
Central to use of electrochemiluminesence methodology as a
measuring system for analytes was the recognition that
.beta.-lactamase can efficiently hydrolyze Ru(bpy).sub.3.sup.2+
substituted penicillins. The penicillins, Ru-Amp and Ru-APA, are
only very weakly electrochemiluminescent but when they are
hydrolyzed by .beta.-lactamase they become strongly
electrochemiluminescent.
Various assay formats can be employed in the practice of the
invention as will be apparent to those skilled in the art. These
include a sandwich assay using, for example, magnetic beads or
other solid support such as carbon fibrils, a competitive assay
using antigen conjugated to free .beta.-lactamase, a competitive
assay where the .beta.-lactamase is a recombinant protein
containing a segment that is bound by an antibody that also binds
the chosen analyte wherein the enzyme is inactivated by antibody
binding, and ELISA where .beta.-lactamase is a reporter on a
secondary antibody. The Immunoassay Handbook, D. Wild, Ed. (1994)
Stockton Press, New York.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows hydrolysis of Ru-AMP and Ru-APA by
.beta.-lactamase.
FIG. 2 shows the synthesis of Ru-AMP.
FIG. 3 shows the synthesis of Ru-APA.
FIG. 4 shows the mass spectrum of the ammonium hexafluorophosphate
salt of Ru-APA.
FIG. 5 shows the proton NMR spectrum of the ammonium
hexafluorophosphate salt of Ru-APA.
FIG. 6 shows the structures of five specific .beta.-lactams.
FIG. 7 shows the hydrolysis by NaOH or by .beta.-lactamase enzyme
of Ru-AMP (left side) and of Ru-APA (right side).
FIG. 8 shows the comparison of measured ECL for a series of
different samples.
FIG. 9 shows the comparison of measured ECL for a series of
different samples.
FIG. 10 shows the effect of unhydrolyzed (closed circles) and
hydrolyzed (open circles) Ru-AMP concentration on the measured
ECL.
FIG. 11 shows the comparison of measured ECL for a series of
different samples.
FIG. 12 shows the effect of unhydrolyzed (closed circles) and
hydrolyzed (open circles) Ru-APA concentration on the measured
ECL.
FIG. 13 shows the comparison of measured ECL for a series of
different samples.
FIG. 14 illustrates an ECL enzyme immunoassay. Various
concentrations of an analyte, RT1 hapten, were immobilized in a
96-well plate. To the plate was added either an antibody-enzyme
conjugate (anti-RT1 antibody covalently coupled to a
.beta.-lactamase enzyme) (Line 1) or non-conjugated antibody or
enzyme (Lines 2-4). Following washing to remove protein that did
not bind to the analyte, the .beta.-lactamase substrate, Pen G, was
added. After incubation to allow any .beta.-lactamase in the plate
to hydrolyze the Pen G, the solutions were withdrawn, mixed with
Ru(bpy).sub.3.sup.2+, and ECL was read in an ECL Analyzer. Line 1
shows the results with the antibody-enzyme conjugate. Lines 2-4
show the results using unconjugated antibody or enzyme.
DETAILED DESCRIPTION OF THE INVENTION
The preferred method of measuring analyte using the
electrochemiluminescence based immunoassay is by the following
sequential steps: 1. In an analyte-containing solution, admix a
magnetic bead-immobilized anti-analyte antibody with a
.beta.-lactamase anti-analyte antibody conjugate. 2. After allowing
antibodies to bind to analyte to create an
antibody-analyte-antibody "sandwich", immobilize the beads with a
magnet, wash extensively to remove non-analyte interfering
molecules and unbound .beta.-lactamase anti-analyte antibody
conjugate. 3. Add ECL-labeled substrate to beads, allow the enzyme
to react, the optimum reaction time being determined by the
expected concentration of the analyte, and withdraw the
supernatant, with no beads. 4. Measure the electrochemiluminescence
of the supernatant and compare it to a standard curve of hydrolyzed
ECL-labeled substrate concentration vs. electrochemiluminescence.
The measurement can be carried out on an ORIGEN.RTM. Analyzer by
following the instructions in the Operators Manual therefor,
available from IGEN, Inc., 16020 Industrial Drive, Gaithersburg,
Md. 20877 U.S.A.
According to the invention, an ECL detectant such as
Ru(bpy).sub.3.sup.2+ is substituted on a substrate such as an
antibiotic, peptide or NADH. An enzyme labeled anti-analyte also is
prepared using .beta.-lactamase. When the ECL substituted substrate
is placed in the presence of the .beta.-lactamase-labeled antibody,
the substrate is catalytically hydrolyzed forming the excited state
of the detectant, Ru(bpy).sub.3.sup.2+ *, in substantial
quantities. The excited state decays to the ground state through a
normal fluorescence mechanism, emitting a photon having a
wavelength of 620 nm.
Organic compounds which are ECL detectants include, for example,
rubrene and 9,10-diphenyl anthracene. Many organometallic compounds
also are ECL detectants, and the most preferable are Ru-containing
compounds, such as ruthenium II tris-bipyridine chelate, and
Os-containing compounds. Detectants useful in the presently
disclosed invention are described in U.S. Pat. No. 5,310,687, the
contents of which are incorporated herein by reference.
These detectants are stable for long periods. In addition, the
detectants are safe and relatively inexpensive. They give a highly
characteristic signal and do not occur in nature. Measurements
based on luminescence of such detectants are sensitive, fast,
reproducible and utilize simple instrumentation. The signal is
generated repeatedly by each molecule of the detectant, thereby
enhancing the sensitivity with which they may be detected. The
preferred electrochemiluminescent detectants of the present
invention are conveniently referred to herein as
Ru(bpy).sub.3.sup.2+. Various amounts of this detectant, or its
equivalent, may be employed. These detectants also have the
advantage that they can be used directly in a biological sample
without pretreatment of the sample.
The energy necessary for formation of the excited state arises from
the hydrolysis of .beta.-lactam or peptide or by reduction of
NAD.sup.+ to NADH. The excited-state Ru(bpy).sub.3.sup.2+ * decays
through a normal fluorescence mechanism, emitting a photon at 620
nm.
Quantification of the Ru(bpy).sub.3.sup.2+ detectant can be readily
automated with relatively uncomplicated instrumentation. The heart
of the instrument is the electrochemical flow cell, containing the
working electrodes and counter electrodes for initiation of the ECL
reaction. Both of the electrodes are preferably fabricated from
gold, but other materials have been used with various degrees of
success. A potentiostat is used to apply various voltage waveforms
to the electrodes, and a single photomultiplier tube (PMT) is used
to detect the light emitted during the ECL reaction. An Ag/AgCl
reference electrode is placed in the fluid path downstream from the
flow cell, and a peristaltic pump is used to draw various fluids
through the flow cell. In a typical sequence, the assay fluid is
drawn from a test tube into the flow cell and the detectant is
quantified by applying a ramp voltage to the electrodes and
measuring the emitted light. After the measurement, a high pH
cleaning solution is drawn into the cell for an electrochemical
cleaning procedure. A conditioning solution is then drawn into the
cell, and a voltage waveform is applied that leaves the surfaces of
the electrodes in a highly reproducible state, ready for the next
measurement cycle.
The ECL reaction can be efficiently initiated by many different
voltage waveforms. Measurements of the working electrode current
and the ECL intensity can be induced, for example, by the
application of a triangle wave to the electrodes. The applied
voltage as shown is actually the voltage measured at the Ag/AgCl
reference electrode and includes the effects of a significant
uncompensated resistance. Consequently, the actual voltage applied
at the working electrode is substantially less than that depicted.
The triangle waveform rises from 565 to 2800 millivolts (mV) at a
rate of 750 millivolts per second (mV/s) and then decreases at the
same rate to 1000 mV. Oxidation of both the .beta.-lactam substrate
and Ru(bpy).sub.3.sup.2+ becomes evident when the applied voltage
reaches 1100 mV and produces a luminescence. The intensity of the
luminescence increases with the applied voltage until the substrate
at the surface of the electrode is depleted, resulting in decreased
intensity. The intensity of the observed luminescence is great
enough that it can easily be measured with conventional
photomultipliers operating either in photon-counting or current
modes.
The preferred method of measuring analyte using the
electrochemiluminescence based immunoassay is by the following
sequential steps: 1. In an analyte-containing solution, admix a
magnetic bead-immobilized anti-analyte antibody with a
.beta.-lactamase anti-analyte antibody conjugate. 2. After allowing
antibodies to bind to analyte to create an
antibody-analyte-antibody "sandwich", immobilize the beads with a
magnet, wash extensively to remove non-analyte interfering
molecules and unbound .beta.-lactamase anti-analyte antibody
conjugate. 3. Add ECL-labeled substrate to beads, allow the enzyme
to react, the optimum reaction time being determined by the
expected concentration of the analyte, and withdraw the
supernatant, with no beads. 4. Measure the electrochemiluminescence
of the supernatant and compare it to a standard curve of hydrolyzed
ECL-labeled substrate concentration vs. electrochemiluminescence.
The measurement can be carried out using established procedures on
the ORIGEN.RTM. Analyzer.
The sample to which the .beta.-lactam of interest has been added is
then placed in a measuring cell to obtain an initial reading.
Typically the .beta.-lactam of interest is added in concentrations
between 10 micromolar and 1.0 millimolar. The
electrochemiluminescent detectant is typically present at
10-.sup.-6 M concentrations (range 1-15 .mu.M). The sample
containing cell is then incubated for a sufficient period of time
to insure that .beta.-lactamase catalyzed hydrolysis can occur if
the enzyme is present. This period of time typically varies between
5 minutes and 2 hours. Longer and shorter periods of time are
possible depending on sample and reagent concentrations. Since all
that is involved is empirical parameters, their values can be
determined using conventional techniques.
After incubation occurs, a second reading is taken. The difference
in readings, if any, correlates with .beta.-lactamase activity
present in the sample. See FIG. 2 in this regard.
Accordingly, the apparatus and methodology suitable for the
performance of the process of this invention include, as noted
earlier, those shown in U.S. Pat. Nos. 5,068,088, 5,061,455,
5,093,268, and 5,147,806 and 5,221,605 which patents are expressly
incorporated herein by reference. In addition,
electrochemiluminesence molecules for use in the measuring system
as detectants include those bidentate aromatic heterocyclic
nitrogen-containing ligands of ruthenium and osmium described in
U.S. Pat. No. 5,310,687, which patent has been expressly
incorporated herein by reference.
Reagent kits containing the materials necessary for the performance
of the assays can be assembled to facilitate handling, and foster
standardization. Materials to be included in the kit may vary
depending on the ultimate purpose. Typically the kit would include
the electrochemiluminescent detectant, necessary buffers, and
standards. The standards can be chemical reagents or data
(empirical) in printed or electronic form necessary for the
calibration needed for performance of the assay.
EXAMPLES
Notwithstanding the previous detailed description of the present
invention, applicants provide below specific examples solely for
purposes of illustration and as an aid to understanding the
invention. The examples are both nonlimiting and nonexclusive.
Accordingly, the scope of applicants' invention as set forth in the
appended claims is to be determined in light of the teachings of
the entire specification.
Example 1
Preparation of Ru(bpy).sub.3.sup.+2 -labeled .beta.-lactam
Antibiotics
(a) Preparation of Ru(bpy).sub.3.sup.+2 -labeled
6-aminopenicillanic Acid ("Ru-APA")
Ru(bpy).sub.3.sup.+2 -NHS ester (15 mg) (IGEN, Inc., Rockville,
Md., USA) in acetonitrile (250 .mu.L) was mixed with
6-aminopenicillanic acid (12.4 mg) in 0.2 M sodium bicarbonate, pH
8.0 (350 .mu.L) and the reaction was allowed to proceed at room
temperature for 2 hours (FIG. 3). Ru-APA was purified with a Waters
HPLC system (Milford, Mass., USA) equipped with a PROGEL.TM.-TSK
CM-5PW column (7.5 cm.times.7.5 mm) (Supelco, Inc., Bellefonte,
Pa., USA) using a 1.0 mL/minute, 20-minute linear gradient from
20-100 mM sodium phosphate, pH 7.0. Substrate was quantitated
spectrophotometrically by measuring the absorbance of the ruthenium
complex (the molar extinction coefficient at 453 nm is 13,700
M.sup.-1 cm.sup.-1).
(b) Preparation of Ru(bpy).sub.3.sup.+2 -labeled Ampicillin
("Ru-AMP")
Ru(bpy).sub.3.sup.+2 -NHS ester (15.1) mg in acetonitrile (250
.mu.L) was mixed with ampicillin (29.1 mg) in 0.2 M sodium
bicarbonate, pH 8.0 (250 .mu.L) and the reaction was allowed to
proceed at room temperature for 2 hours (FIG. 2). Ru-AMP was
purified using a Waters HPLC system (Milford, Mass., USA) equipped
with a PROGEL.TM.-TSJ CM-15PW column (7.5 cm.times.7.5 mm)
(Supelco, Inc., Bellefonte, Pa., USA) using a 1.0 mL/minute,
15-minute linear gradient from 20-180 mM sodium phosphate, pH 7.0.
Substrate was quantitated spectrophotomerically by measuring the
absorbance of the ruthenium complex (the molar extinction
coefficient at 453 nm is 13,700 M.sup.-1 cm.sup.-1). Following
formation of the amnmonium hexafluorophosphate salt, the structure
and purity of Ru-AMP was confirmed by mass spectroscopy and proton
NMR (FIGS. 4-5).
(c) Preparation of other Ru(bpy).sub.3.sup.+2 labeled
.beta.-lactmans
Other .beta.-lactmans, such as 7-aminocephalosporanic acid, that
have a primary amine in their structures can also react with
Ru(bpy).sub.3.sup.+2 -NHS ester to form similar conjugates as
described above. The reaction and purification conditions will be
similar potentially difering somewhat in ways solvable by one
skilled in the art . FIG. 6 shows the structure of 5 specific
.beta.-lactams.
Example 2
ECL Assay of Ru-AMP Hydrolysis
Experiments were performed to compare the ECL properties of Ru-AMP
(conjugated) with Ru(bpy).sub.3.sup.+2 and ampicillin mixtures
(nonconjugated). ECL properties were compared both before and after
NaOH and enzymatic hydrolysis (FIG. 7).
Ru-AMP was found to be a very good substrate of .beta.-lactamase.
Hydrolysis of Ru-AMP (33 .mu.M) by .beta.-lactamase I from Bacillus
cereus (0.3 nM) was monitored spectrophotometrically at 240 nm
using a Hitachi U3200 spectrophotometer (Danbury, Conn., USA) at
25.0.degree. C. in 0.1 M sodium phosphate, pH 7.0. Half-time
(t.sub.1/2) analysis gave a k.sub.cat /K.sub.m for enzymatic
hydrolysis of Ru-AMP of 3.9.times.10.sup.8 min.sup.-1 M.sup.-1.
The ECL properties of equimolar mixtures of Ru(bpy).sub.3.sup.+2
and ampicillin (hydrolyzed or unhydrolyzed) were compared to the
same concentration of the Ru-AMP conjugate (hydrolyzed or
unhydrolyzed). In separate experiments, ampicillin and Ru-AMP were
hydrolyzed by either 50 mM NaOH (base hydrolysis) or 347 nM
.beta.-lactam I from Bacillus cereus (enzyme hydrolysis). For base
hydrolysis, 50 .mu.L of 5 M NaOH were added to 1.0 mL solutions of
deionized water containing either 30.1 .mu.M Ru-AMP or a mixture of
30 .mu.M ampicillin and 30 .mu.M Ru(bpy).sub.3.sup.+2. Following 30
minute incubations, the solutions were neutralized with 50 .mu.L of
5 M HCl. For the unhydrolyzed counterpart experiments, 50 .mu.L of
5 M H.sub.2 O were added to solutions of either 30.1 .mu.M Ru-AMP
or a mixture containing 30.0 .mu.M ampicillin and 30.0 .mu.M
Ru(bpy).sub.3.sup.+2. Following 30 minute incubations, 50 .mu.L of
5 M NaCl was added to these solutions. The results shown in FIG. 8
demonstrate: (1) that ampicillin hydrolysis by either NaOH or
.beta.-lactamase causes an increase in the ECL of the mixtures; and
(2) that the increase in the ECL caused by the hydrolysis is
dramatically greater when the light-emitting ruthenium complex is
covalently linked to ampicillin. With base hydrolysis, ECL
increased 1.5-fold when ampicillin was hydrolyzed in a mixture of
ampicillin and Ru(bpy).sub.3.sup.+2, while ECL increased 5.2-fold
when Ru-AMP was hydrolyzed. Similar results were obtained in enzyme
hydrolysis: ECL increased 2.1-fold when ampicillin was hydrolyzed
in a mixture of ampicillin and Ru(bpy).sub.3.sup.+2, while ECL
increased 9.8-fold upon hydrolysis of Ru-AMP. The data establishing
these conclusions is found in FIG. 8 which shows the experimentally
measured electrochemiluminescence of (from left to right):
Ru(bpy).sub.3.sup.+2 alone; Ru(bpy).sub.3.sup.+2 plus unhydrolyzed
ampicillin; Ru(bpy).sub.3.sup.+2 plus NaOH-hydrolyzed ampicillin;
unhydrolyzed Ru-AMP; NaOH-hydrolyzed Ru-AMP; Ru(bpy).sub.3.sup.+2
plus unhydrolyzed ampicillin; Ru(bpy).sub.3.sup.+2 plus
.beta.-lactamase-hydrolyzed ampicillin; unhydrolyzed Ru-AMP; and
.beta.-lactamase-hydrolyzed Ru-AMP.
This work was confirmed in a second experiment using enzyme
hydrolysis which differed in that the incubating time with enzyme
was lengthened from 30 to 60 minutes (FIG. 9). Here, enzyme
hydrolysis caused a 2.5-fold increase in ECL when ampicillin and
Ru(bpy).sub.3.sup.+2 were conjugated and an 11.1-fold increase in
ECL when the Ru-AMP conjugate was hydrolyzed. The data establishing
these conclusions is found in FIG. 9 which shows the experimentally
measured luminescence of (from left to right): Ru(bpy).sub.3.sup.+2
alone; Ru(bpy).sub.3.sup.+2 plus unhydrolyzed ampicillin;
Ru(bpy).sub.3.sup.+2 plus .beta.-lactamase-hydrolyzed ampicillin;
unhydrolyzed Ru-AMP; and .beta.-lactamase-hydrolyzed Ru-AMP.
These results show that Ru(bpy).sub.3.sup.+2 -conjugation caused
intramolecular effects that dramatically increase the
experimentally measured luminescence when the .beta.-lactam ring is
hydrolyzed.
FIG. 10 shows that low concentrations of Ru-AMP can be detected by
hydrolysis. The lower limit of detection was found to be 50 nM (464
relative ECL counts for hydrolyzed Ru-AMP versus an average
instrument reading of -152 relative counts for unhydrolyzed
Ru-AMP). This compares favorably to the lower limit for detection
of (unconjugated) ampicillin hydrolysis which was 5000 nM.
Example 3
ECL Assay of RU-APA Hydrolysis
It was thought that Ru-APA might have different ECL properties
(before and after hydrolysis) from those of Ru-AMP. The differences
would be a consequence of the structural differences between APA
and AMP, especially the difference in distance between the
.beta.-lactam ring and the primary amino group used to conjugate
Ru(bpy).sub.3.sup.+2 -NHS ester (FIG. 7). In Ru-AMP, the
.beta.-lactam ring is three bond lengths farther from the amino
group than in Ru-APA. Specifically, hydrolysis of Ru-APA (or other
.beta.-lactam conjugates) may be more or less sensitively detected
by ECL than Ru-AMP hydrolysis.
The ECL properties of the Ru-APA conjugate were compared with those
of the mixtures of unconjugated Ru(bpy).sub.3.sup.+2 and 6-APA. ECL
properties were compared before and after NaOH and enzymatic
hydrolysis. The data was then compared to the results of similar
experiments with Ru-AMP described in Example 2.
Ru-APA was found to be a very good substrate of .beta.-lactamase.
Hydrolysis of Ru-APA (23 .mu.M) by .beta.-lactamase I from Bacillus
cereus (0.6 nM) was monitored spectrophotometrically at 240 nm
using a Hitachi U3200 spectrophotometer (Danbury, Conn., USA) at
25.0.degree. C. in 0.1 M sodium phosphate, pH 7.0. Half-time
(t.sub.1/2) analysis gave a k.sub.cat /K.sub.m for enzymatic
hydrolysis of Ru-APA of 9.8.times.10.sup.7 min.sup.-1 M.sup.-1.
The ECL properties of equimolar mixtures of Ru(bpy).sub.3.sup.+2
and ampicillin (hydrolyzed or unhydrolyzed) were compared with the
same concentration of the Ru-APA conjugate (hydrolyzed or
unhydrolyzed). In separate experiments, 6-APA and Ru-APA were
hydrolyzed by either 50 mM NaOH (base hydrolysis) or 3.8 .mu.M
.beta.-lactamase I from Bacillus cereus (enzyme hydrolysis).
For base hydrolysis, 50 mL of 5 M NaOH were added to 1.0 mL
solutions of deionized water containing either 23.0 .mu.M Ru-APA or
a mixture of 23.0 .mu.M APA and 23.0 .mu.M Ru(bpy).sub.3.sup.+2.
Following 30 minute incubations, the solutions were neutralized
with 50 .mu.L of 5 M HCl. For unhydrolyzed counterpart experiments,
50 .mu.L of 5 M H.sub.2 O were added to solutions of either 23.0
.mu.M Ru-APA or a mixture of 23.0 .mu.M APA and 23.0 .mu.M
Ru(bpy).sub.3.sup.+2. Following 60 minute incubations, 50 .mu.L of
5 M NaCl was added to these solutions. The results shown in FIG. 11
demonstrate: (1) that 6-APA (conjugated or nonconjugated)
hydrolysis by either NaOH or .beta.-lactamase causes an increase in
the ECL; and (2) that the increase in the ECL caused by the
hydrolysis is dramatically greater when the light-emitting
ruthenium complex is covalently coupled to 6-APA. With base
hydrolysis, ECL increased 1.9-fold when 6-APA (nonconjugated) in a
mixture of 6-APA and Ru(bpy).sub.3.sup.+2, was hydrolyzed, while
ECL increased 13.2-fold when Ru-APA (conjugated) was hydrolyzed.
Similarly with enzyme hydrolysis, ECL increased 1.4-fold when 6-APA
(nonconjugated) in a mixture of 6-APA and Ru(bpy).sub.3.sup.+2 was
hydrolyzed, while ECL increased 31.8-fold when Ru-APA (conjugated)
was hydrolyzed. The data establishing these conclusions is found in
FIG. 11 which shows the experimentally measured luminescence of
(from left to right): Ru(bpy).sub.3.sup.+2 alone;
Ru(bpy).sub.3.sup.+2 plus unhydrolyzed 6-APA; Ru(bpy).sub.3.sup.+2
plus NaOH-hydrolyzed 6-APA; unhydrolyzed Ru-APA; NaOH-hydrolyzed
Ru-APA; Ru(bpy).sub.3.sup.+2 plus unhydrolyzed 6-APA;
Ru(bpy).sub.3.sup.+2 plus .beta.-lactamase-hydrolyzed 6-APA;
unhydrolyzed Ru-APA; and .beta.-lactamase-hydrolyzed APA.
This work clearly demonstrates that conjugation of the 6-APA and
the electrochemiluminescent ruthenium complex result in
intramolecular effects that increase the electrochemiluminescence
when the .beta.-lactam ring is hydrolyzed. Moreover, comparison
with the results described in Example 2 for the ampicillin
conjugate show that hydrolysis of Ru-APA results in a much greater
electrochemiluminescence signal than hydrolysis of Ru-AMP. Because
the ruthenium atom is closer to the .beta.-lactam ring in Ru-APA
than in Ru-AMP, these results indicate that there may be a critical
effect of the distance between the ruthenium complex and the
.beta.-lactam ring. Other, as-yet untested
.beta.-lactam-Ru(bpy).sub.3.sup.+2 conjugates may give an even more
dramatic change in the electrochemiluminescence upon .beta.-lactam
hydrolysis.
FIG. 12 shows that the hydrolysis of very low concentrations of
Ru-APA can be detected by ECL. More specifically, FIG. 12 shows the
effect of unhydrolyzed (closed circles) and hydrolyzed (open
circles) Ru-APA concentration on the experimentally measured
electrochemiluminescence. The lower limit of detection was found to
be 50 nM (an instrument reading of -33 relative ECL counts for
hydrolyzed Ru-APA versus an average of -648 relative ECL counts for
unhydrolyzed Ru-APA (conjugated)). This compares favorably to the
lower limit for detection of (unconjugated) ampicillin hydrolysis
which was 50 .mu.M (in the presence of 10 .mu.M
Ru(bpy).sub.3.sup.+2).
An experiment was performed to quantitate the advantage of
conjugating a .beta.-lactam to the ECL label, Ru(bpy).sub.3.sup.+2.
The increase in ECL upon hydrolysis of 10 .mu.M Ru-APA was compared
to an ECL standard curve in which various concentrations of 6-APA
(nonconjugated) were hydrolyzed in the presence of 10 .mu.M
Ru(bpy).sub.3.sup.+2. By extrapolation of the 6-APA standard curve,
the results (FIG. 13) demonstrates that the ECL change upon
hydrolysis of 10 .mu.M Ru-APA (conjugated) is equivalent to the ECL
change in the hydrolysis of 1250 .mu.M 6-APA (nonconjugated) in the
presence of 10 .mu.M Ru(bpy).sub.3.sup.+2. This demonstrates that
conjugation of Ru(bpy).sub.3.sup.+2 and 6-APA results in a 125-fold
increase in the ECL change seen during 6-APA hydrolysis. The data
establishing these conclusions is found at FIG. 13 which shows a
comparison of electrochemiluminescence effects of Ru-APA
(conjugated) to Ru(bpy).sub.3.sup.+2 plus 6-APA (unconjugated).
Triangles represent the electrochemiluminescence of 10 .mu.M
unhydrolyzed (open triangles) and hydrolyzed (closed triangles)
Ru-APA. Circles represent the electrochemiluminescence effects of
unhydrolyzed (closed circles) and hydrolyzed (open circles) 6-APA
(0-1000 .mu.M) in the presence of 10 .mu.M Ru(bpy).sub.3.sup.+2.
Extrapolation in FIG. 13 indicates the electrochemiluminescence
change upon hydrolysis of 10 .mu.M Ru-APA is equivalent to the
electrochemiluminescence change upon hydrolysis of 1250 .mu.M free
6-APA in the presence of 10 .mu.M Ru(bpy).sub.3.sup.+2.
Example 4
Preparation of an Antibody-.beta.-Lactamase Conjugate
Antibody-B-Lactamase conjugates have been previously prepared
(Yolken et al., J. Immunol. Meth. 73 (1984) 109-123; Svensson et
al., Bioconj. Chem. 5 (1994) 262-267). Conjugates are generally
prepared using commercially available bifunctional crosslinking
agents such as Sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl]
cyclohexane-1-carboxylate), which was used here. Other methods of
covalently linking two proteins have been established and could
also be used. Any method is satisfactory as long as the antibody
and the enzyme remain biologically active after conjugation.
.beta.-Lactamase (3.7 mg) was dissolved in 0.500 mL phosphate
buffered saline (PBS). Sulfo-SMCC (5 mg) was dissolved in 1.500 mL
PBS. The solutions of .beta.-lactamase and Sulfo-SMCC were mixed
and allowed to react for 45 min. at room temperature.
A monoclonal antibody raised against the hapten RT1 (5 mg) was
buffer-exchanged into PBS using a Centricon 30 concentrator
(Amicon). Dithiothreitol (DTT, 5 mg) was dissolved in PBS, then
mixed with the anti-RT1 antibody to give a total volume of 1.300
mL. The mixture is incubated for 30 min. at room temperature to
allow DTT to reduce the disulfide bonds of RT1.
The proteins in the two reaction mixtures described above were
desalted using SEPHADEX G-25M PD-10 columns (Pharmacia) which had
been prequilibrated with PBS. The recovered proteins were
quantitated spectrophotometrically at 280 nm. The yields were found
to be 1.0 mg .beta.-lactamase and 3.1 mg antibody. The protein
solutions were then mixed giving a 1.5:1.0 molar ratio of
.beta.-lactamase to antibody. The protein solution was rotated at
420.degree. C. for 22 hr. to allow the enzyme-antibody conjugate to
form. Following the reaction, the mixture was chromatographed on a
Sephacryl S-300 column (Pharmacia). Three major protein peaks were
obtained. Because the chromatographic separation was by size, the
first peak to elute from the column was expected to be the enyme
antibody conjugate.
Example 5
ECL Enzyme Immunoassay
An ECL immunoassay using a .beta.-lactamase-antibody conjugate can
be carried out either with an unconjugated mixture of
Ru(bpy).sub.3.sup.+2 and a .beta.-lactam antibiotic (such as APA or
Pen G) or, preferably, with a Ru(bpy).sub.3.sup.2+ -.beta.-lactam
conjugate (such as Ru-APA). Using a conjugated ECL substrate system
is preferred because hydrolysis of Ru(bpy).sub.3.sup.2+ -labelled
substrates is much more sensitively detected by ECL than mixtures
of the substrate and Ru(bpy).sub.3.sup.2+ and the .beta.-lactamase
substrate, Pen G.
Here, an ECL enzyme immunoassay was tested using an antibody-enzyme
conjugate (anti-RT1 antibody linked to .beta.-lactamase as
described in Example 4). The presence of the analyte was reported
by the .beta.-lactamase portion of the conjugate, which hydrolyzed
the penicillin, Pen G, which is turn caused Ru(bpy).sub.3.sup.2+ to
emit light by elecrochemiluminescence. The assay was performed in a
96-well plate and ECL was measured by transferring the contents of
the wells into test tubes which were read in an ORIGEN.RTM.
Analyzer.
The analyte (the RT1 hapten conjugated to Bovine Serum Albumin
(BSA)) was incubated for 2 hours at 37.degree. C. in a 96-well
plate at 0, 0.2, 2.0, and 10.0 .mu.g/ml to allow it to adhere to
the plate. The plate was then washed three times with PBS. To each
well was then added 200 .mu.L of 3% BSA in PBS and the plate was
incubated for about 1 hour at 37.degree. C. To each well was added
50 .mu.L of chromatography fractions from Example 4. The fractions
from the first protein peak to elute are suspected to be the
antibody-enzyme conjugate while the fractions from the later
eluting protein peaks are suspected to be either free antibody or
free enzyme, neither of which should give an ECL signal in this
experiment. The plate was incubated overnight at 4.degree. C. to
allow the antibody-enzyme conjugate to bind to the analyte. The
plate was then washed three times with PBS containing 0.05% Tween.
To each well was added 75 .mu.L of 10 mM Pen G and the plate was
incubated for 30 min. at room temperature to allow any
.beta.-lactamase present to hydrolyze the Pen G. Following the
incubation period, 25 .mu.L was transferred from each well to test
tubes. To each tube was added 25 .mu.L of 120 .mu.M
Ru(bpy).sub.3.sup.2+ and 250 .mu.L of 0.1 M sodium phosphate, pH
7.0. ECL of the mixtures was then read in an ORIGEN.RTM.
Analyzer.
The results of the ECL enzyme immunoassay are shown in FIG. 14. The
protein used in Line 1 was the expected antibody-enzyme conjugate.
As can be seen in FIG. 14, the ECL counts in Line 1 increase with
increasing analyte concentration. This indicates that the
antibody-enzyme conjugate bound to the analyte and hydrolyzed Pen G
to a form which promotes Ru(bpy).sub.3.sup.2+ ECL. Even the lowest
concentration of analyte tested, 0.2 .mu.g/mL, was detectable. The
other lines (2-4) show other chromatographic fractions
representing, presumably, free antibody and free enzyme. These
lines, which can be considered control experiments, show little
increase in ECL with increasing concentrations of analyte. In
summary, the antibody-enzyme conjugate was used in an enzyme
immunoassay to sensitively detect an analyte using an unconjugated
mixture of Pen G and Ru(bpy).sub.3.sup.2+. Because the
Ru(bpy).sub.3.sup.2+ -.beta. lactam conjugated substrate is much
more sensitive in detecting .beta.-lactam hydrolysis by ECL than a
mixture of Ru(bpy).sub.3.sup.2+ and .beta.-lactam, the results
described here can probably be vastly improved by using a
conjugated substrate.
* * * * *